Fas is a polypeptide that exists in the surfaces of a variety of cells and is considered to be deeply concerned with the apoptosis of cells. The apoptosis is a form of death of cells that is distinguished from the so-called necrosis of cells, and is observed at the time of death of various cells such as of embryogenesis, metamorphosis, endocrine-dependent tissue atrophy and turnover of normal tissues [Wyllie et al. Int. Rev. Cytol. 68, 251-306, 1980; Walker et al. Meth. Achiev. Exp. Pathol. 13, 18-54, 1988; Schmidt et al. Proc. Natl. Acad. Sci. USA 83, 1881-1885, 1986; Ucker et al. Nature 327, 62-64, 1987; Smith et al. Nature 337, 181-184, 1989, Williams et al. Nature 343, 76-79, 1990]. The following features have been pointed out as a result of the morphological and biochemical analyses of cells at the apoptosis:
The apoptosis is accompanied by condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, extensive degradation of chromosomal DNA (into oligomers of about 180 base pair units), and formation of apoptotic bleb [Wyllie et al. 1980 (mentioned above)]. The apoptosis is a physiologically and medically interesting phenomenon because it is a form associated with the death of immunocytes such as thymocytes and the extinction of the tumor cells.
In regression of tumor (alleviation of tumor), in general, the apoptosis mediates the death of target cells by interaction with natural killer cells or cytotoxic T lymphocytes [Duke et al. Proc. Natil. Acad. Sci. USA 80, 6361-6365, 1983; Schmidt et al, 1986 ibid.; Ucker, 1987 (mentioned above)], or by tumor necrosis factor-α (TNF-α) or its related cytokine lymphotoxin (TNF-β) against the target cells [Schmidt et al, 1986 (mentioned above); Dealtry et al. Eur. J. Immunol., 17, 689-693, 1987; Larrick and Wright, FASEB J. 4, 3215-3223, 1990].
With regard to the relationship between the Fas antigen and the apoptosis, the present inventors have previously disclosed that the mouse monoclonal antibody against the human Fas antigen has a cytolytic activity on human cells expressing the Fas antigen while it does not act upon mouse cells [Yonehara et al. J. Exp. Med. 169, 1747-1756, 1989]. It has also been disclosed by Trauth et al. that the anti-Apo-I antibody has effects analogous to those of the anti-Fas antibody [Science 245, 301-305, 1989].
In a recent study by the present inventors, furthermore, it has been found that cells infected with human immunodeficiecy virus (HIV) are more sensitive to the cytocidal activity of the anti-Fas monoclonal antibody than uninfected cells [Kobayashi et al. Proc. Natl. Acad. Sci. USA 87, 9620-9621, 1990]. However, it is still not clear whether the expression of the Fas antigen that is predominant in the infected cells is actually induced by infection with HIV or by a general transformation. It is also considered potential to specifically drive the HIV-infected cells into apoptosis by using a monoclonal antibody specific to Fas antigen.
The present inventors have further discovered that the treatment of human colon carcinoma HT-29 cells with interferon-γ (INF-γ) induces the Fas antigen on the cell surface, and renders the tumor cells more susceptible to the cytotoxic activity of the anti-Fas antibody (Yonehara et al, 1989 (mentioned above)).
As described above, it has been pointed out that the Fas antigen is closely related to the apoptosis but numerous points remain not clarified. Therefore, it is physiologically and pathologically meaningful to disclose the entire structure of the Fas antigen and to clarify its function. It is further considered that various monoclonal antibodies that specifically reacts with Fas may be easily obtained if the structure of the Fas antigen is disclosed, and used in treating diseases associated with HIV infection and malignant tumors to be cured.
Therefore, it is physilogically and pathologically very advantageous to clarify the main body of Fas antigen, to clarify its complete structure and to clarify its function. Furthermore, if the Fas antigen is obtained in large amounts in pure form, it will become possible to more clearly analyze its structure and functions. By utilizing the knowledge related to the thus clarified structure of Fas antigen, it will still become possible to study the Fas antigen analogs by modifying them as well as to utilize in large amounts only those portions essential to the expression of the functions.
With the structure of the Fas antigen being clarified, furthermore, it will become possible to obtain various monoclonal antibodies that specifically reacts with Fas as well as to obtain various ligands, agonists and antagonists related to Fas, and to develop studies with regard to their effects upon the cells and relationships of the structure and activities thereof.
In order to accomplish the above object, it is essential to establish means capable of supplying Fas polypeptides in sufficient amounts. In recent years, a recombinant DNA technology has been utilized as a method for preparing physiologically active substance. In order to prepare the Fas antigen by utilizing the above technology, however, it is necessary to isolate DNA that encodes Fas proteins followed by cloning.